Peptoid Coatings For Neural Stem Cell Differentiation Poster 2017 2.p… · Joshua Corbitt, Jesse...
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Peptoid Coatings For Neural Stem Cell Differentiation Joshua Corbitt, Jesse Roberts, Dr. German R. Perez Bakovic, Dr. Shannon Servoss, and Dr. Michael Ceballos Ralph E. Martin Department of Chemical Engineering, University of Arkansas Abstract Neurons differentiated from human neural stem cells have shown promise for the purposes of tissue engineering and regenerative medicine. The chemical signals and physical characteristics provided by the extracellular matrix (ECM) are crucial in the differentiation and proliferation of neural stem cells. Experiments crafted to determine what features of the ECM affect stem cell growth are necessary to further their applications. Promising studies have focused on the development of scaffolds with nanoscale features that can incorporate neural differentiation enhancers. In this work, we have coated surfaces with peptoid microspheres. Peptoids are synthetic poly-n- substituted Glycines designed to mimic peptide bioactivity while remaining protease- resistant. The nature of peptoid synthesis allows the customization of every side chain addition to the backbone of the peptoid. Preliminary studies show that the peptoid coatings enhance cell differentiation into neurons and support neurite outgrowth. This work is being extended to determine the mechanism of action, as well as incorporation of other neural stem cell differentiation modifiers. In this study, immunofluorescent staining was used to characterize cells. Coating morphology was evaluated using SEM and the binding efficacy of specialized linkages was analyzed by ELISA. Fluorescent imaging was captured by the Leica SP 5 Confocal Microscope. Acknowledgements Background Peptoids (Poly-N-Substituted Glycines) Bio-inspired peptidomimetic polymers • Nitrogen attached side-chains Attractive as biomaterials for therapeutic applications • Highly customizable chemistry • Low immunogenicity • Protease resistant Peptoid Synthesis Peptoid Microsphere Formation NH N N N N N N N N N N N NH 2 O O O O O O O O O O O O O N H 3 + O N H 3 + MW: 1919 Da The inclusion of chiral aromatic side chains sterically induces helical structure • Helical peptoids that are partially soluble in water self-assemble into microspheres • Spheres can be tailored varying side chain placement and chemistry Peptoid Coating Depositions Perimetral intensive depositions are indicative of accumulations at the air/liquid interface Surfactants • Preserve a constant contact area • Competitively displace substrates at the air/liquid interface ✓ Lessen perimetral deposition Coating morphology is directly linked to the mode of evaporation Constant contact area is desired for the deposition of uniform coating Submonomer addition method Desired Sequence The Servoss Lab has demonstrated the formation of self-assembling peptoid spheres. Work here describes the controlled deposition of robust and uniform coatings Addition of surfactant (0.05%) Tween improved the overall uniformity of the coatings Spheres size variations based on different peptoid chain lengths: Introduction of Human Neural Stem Cells Neuron stem cells grown in culture flasks from three days after thaw and just before second split (left to right). Conclusions and Future Work To be practical in biosensor applications coatings must withstand multiple conditions The robustness of the coating was assessed under physiological conditions • SEM analysis of the surface reveal no perceptible effects on microsphere morphology ✓ Cultured neural stem cell to differentiation stage • Successfully cultured neural stem cells using sterile procedures • Developed procedure to introduce peptoid coating into the differentiation process ✓ Cells grow in the crevasses of the peptoid substrate. • Z-Stack imaging on the Leica SP 5 Confocal Microscope showed different levels of focus on the cells → Design an affective immunostaining procedure for clear imaging of Cells 18mer 24mer • Center for Advanced Surface Engineering (CASE) Arkansas NSF EPSCoR Program • The Arkansas Statewide Mass Spectrometry Facility • Dr. Joshua Ovila Marceau and Safiya-Hana Belbina • Arkansas Nano & Bio Materials Characterization Facility Cells on day four of differentiation procedure, control on left peptoid microsphere sample on right. Blue stain is DAPI for nucleus, green is beta Tubulin for neurons, and red is GFAP for astrocytes. Images derived from confocal using z-stack imaging to show three-dimensional growth. 40X 40X
Transcript of Peptoid Coatings For Neural Stem Cell Differentiation Poster 2017 2.p… · Joshua Corbitt, Jesse...
Peptoid Coatings For Neural Stem Cell DifferentiationJoshua Corbitt, Jesse Roberts, Dr. German R. Perez Bakovic,
Dr. Shannon Servoss, and Dr. Michael Ceballos Ralph E. Martin Department of Chemical Engineering, University of Arkansas
AbstractNeurons differentiated from human neural stem cells have shown promise for the
purposes of tissue engineering and regenerative medicine. The chemical signals and
physical characteristics provided by the extracellular matrix (ECM) are crucial in the
differentiation and proliferation of neural stem cells. Experiments crafted to determine
what features of the ECM affect stem cell growth are necessary to further their
applications. Promising studies have focused on the development of scaffolds with
nanoscale features that can incorporate neural differentiation enhancers. In this work,
we have coated surfaces with peptoid microspheres. Peptoids are synthetic poly-n-
substituted Glycines designed to mimic peptide bioactivity while remaining protease-
resistant. The nature of peptoid synthesis allows the customization of every side
chain addition to the backbone of the peptoid. Preliminary studies show that the
peptoid coatings enhance cell differentiation into neurons and support neurite
outgrowth. This work is being extended to determine the mechanism of action, as
well as incorporation of other neural stem cell differentiation modifiers. In this study,
immunofluorescent staining was used to characterize cells. Coating morphology was
evaluated using SEM and the binding efficacy of specialized linkages was analyzed
by ELISA. Fluorescent imaging was captured by the Leica SP 5 Confocal
Microscope.
Acknowledgements
Background
Peptoids (Poly-N-Substituted Glycines)
Bio-inspired peptidomimetic polymers
• Nitrogen attached side-chains
Attractive as biomaterials for therapeutic applications
• Highly customizable chemistry
• Low immunogenicity
• Protease resistant
Peptoid Synthesis
Peptoid Microsphere Formation
NN
NN
NN
NN
NN
NN
NH2
O
O
O O
O O O
OO
O
O
O
O
OH
O
OH
O
NH3
+
NH3
+
NHN
NN
NN
NN
NN
NN
NH2
O
O O
O O O
OO
O
O
O
O
O
NH3
+
O
NH3
+
MW: 1919 Da
The inclusion of chiral aromatic side chains sterically induces helical structure
• Helical peptoids that are partially soluble in water self-assemble into microspheres
• Spheres can be tailored varying side chain placement and chemistry
Peptoid Coating Depositions
Perimetral intensive depositions are indicative of
accumulations at the air/liquid interface
Surfactants
• Preserve a constant contact area
• Competitively displace substrates at the air/liquid interface
✓ Lessen perimetral deposition
Coating morphology is directly linked
to the mode of evaporation
Constant contact area is desired for
the deposition of uniform coating
Submonomer addition method
Desired Sequence
The Servoss Lab has demonstrated the formation of self-assembling peptoid spheres.
Work here describes the controlled deposition of robust and uniform coatings
Addition of surfactant (0.05%) Tween improved the overall uniformity of the coatings
Spheres size variations based on different peptoid chain lengths:
Introduction of Human Neural Stem CellsNeuron stem cells grown in culture flasks from three days after thaw and just before
second split (left to right).
Conclusions and Future Work
To be practical in biosensor applications coatings must withstand multiple conditions
The robustness of the coating was assessed under physiological conditions
• SEM analysis of the surface reveal no perceptible effects on microsphere morphology
✓ Cultured neural stem cell to differentiation stage
• Successfully cultured neural stem cells using sterile procedures
• Developed procedure to introduce peptoid coating into the differentiation process
✓ Cells grow in the crevasses of the peptoid substrate.
• Z-Stack imaging on the Leica SP 5 Confocal Microscope showed different levels of
focus on the cells
→ Design an affective immunostaining procedure for clear imaging of Cells
18mer 24mer
• Center for Advanced Surface Engineering (CASE) Arkansas NSF EPSCoR Program
• The Arkansas Statewide Mass Spectrometry Facility
• Dr. Joshua Ovila Marceau and Safiya-Hana Belbina
• Arkansas Nano & Bio Materials Characterization Facility
Cells on day four of differentiation procedure, control on left peptoid microsphere
sample on right. Blue stain is DAPI for nucleus, green is beta Tubulin for neurons, and
red is GFAP for astrocytes.
Images derived from confocal using z-stack imaging to show three-dimensional growth.
40X 40X
